Bat Red Blood Cells Express Nucleic Acid-Sensing Receptors and Bind RNA and DNA

Abstract

RBCs demonstrate immunomodulatory capabilities through the expression of nucleic acid sensors. However, little is known about bat RBCs, and no studies have examined the immune function of bat erythrocytes. In this study, we show that bat RBCs express the nucleic acid-sensing TLRs TLR7 and TLR9 and bind the nucleic acid ligands, ssRNA, and CpG DNA. Collectively, these data suggest that, like human RBCs, bat erythrocytes possess immune function and may be reservoirs for nucleic acids. These findings provide unique insight into bat immunity and may uncover potential mechanisms by which virulent pathogens of humans are concealed in bats.

Figure 1.. Bat RBCs bind nucleic acids.

(A-D) Morphological Characteristics of Bat RBCs. (A) Peripheral smears of RBCs from six bats. (B) Percentage of normal and abnormal RBCs in individual bats. (C) Similar numbers of normally shaped and abnormally shaped RBCs are observed in bats. (D) Percentage of abnormal RBCs in bats. Arrows denote erythrocyte ghosts. (E and F) Imaging Flow Cytometry of CpG-treated Bat RBCs. (E) PBS- and (F) ODN2006-treated RBCs reveal four distinct populations of cells and acquisition of DNA by RBCs. (G and H) Dose-dependent binding of ssRNA (RNA40), CpG ODN 1826 or 2006; contour plots and histograms for one representative bat are shown in (G), and summary statistics for percent positive cells in (H). n=8 bats. (I and J) Competitive inhibition of CpG binding to bat RBCs; contour plots (I) and percent CpG-positive cells and MFI (J) are shown for two bats.

Figure 2.. Bat RBCs contain DNA and RNA and bind pathogen derived RNA

(A) 16S rDNA gene copies associated with RBCs derived from healthy human donors and bats. (B) 18S rRNA content of bat and human RBCs. For (A and B), each dot represents an individual bat or human donor sample,****p<0.0001. (C) qPCR quantitation for ZIKV RNA on RBCs incubated with varying concentrations of ZIKV RNA with PCR of the envelope (E) and non-structural protein 5 (NS5) genes. (D) qPCR for SARS-CoV-2 RNA on RBCs incubated with varying concentrations of SARS-CoV-2 RNA with PCR for two different amplicons for nucleocapsid gene (N1 and N2). n=1 bat. For (C and D), technical replicates are shown for each dose of RNA used. **P<0.01, ****p<0001.

Figure 3.. TLR expression on Bat RBCs.

(A) Intracellular staining for TLR 9, 7, and 3 from three representative bats. (B) Summary statistics, percent positive cells, and MFI for TLRs, n=5, *P<0.05, **P<0.01. (C) Immunofluorescent staining for TLR7 and TLR9, magenta is autofluorescence, and green is TLR staining.